Treatment of tissue specimens

ABSTRACT

Apparatus for treating tissue specimens comprises a stationary outer casing ( 2 ) in the top of which is positioned an annular trough ( 3 ) for holding a liquid. Rotatably mounted in the outer casing ( 2 ) is a central hub ( 5 ) supporting a disc-like lid ( 6 ) the underside of which carries a magnet ( 7 ). In use, the magnets hold, in a detachable manner, tissue specimens ( 12 ) and the trough ( 3 ) is filled with a liquid for treating the specimens. The hub ( 5 ) is capable of vertical translational movement with respect to the casing ( 2 ) to enable the specimens to be lowered into and subsequently lifted out of the trough ( 3 ). A method of treating tissue specimens is also provided.

FIELD OF THE INVENTION

This invention relates to the treatment of tissue specimens,particularly (but not exclusively) prior to imaging of the specimens byoptical projection tomography (OPT), and to uses of optical projectiontomography methods and apparatus.

BACKGROUND TO THE INVENTION

Tissue specimens are normally treated by immersion in one or moreliquids, before the specimens are imaged in OPT apparatus, and theinvention was devised as a way of facilitating this pre-treatment ofspecimens.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided apparatus fortreating tissue specimens by immersion in a liquid, the apparatuscomprising a first structure providing a chamber for holding the liquid,and a second structure including holding means for releasably holdingthe specimens, the first and second structures being relatively moveablein a direction having a vertical component between a first position inwhich the holding means are relatively close to the chamber and in whichthe second structure closes the top of the chamber to enable thespecimens to be immersed in the liquid whilst the latter is protectedfrom the environment, and a second position in which the holding meansare relatively distant from the chamber to enable the specimens to beloaded onto or unloaded from the holding means.

Preferably, the first structure is stationary and the second structureis shiftable vertically with respect to the first structure.

The holding means may include magnets to enable specimens, each providedwith a metal mount, to be detachably retained on the second structure bymagnetic attraction.

The chamber is preferably in the form of an annular trough in which casethe holding means may hold the specimens so that the latter depend fromthe holding means, conveniently at angularly spaced positions around acircle such that the specimens are lowered into the trough as the secondstructure is lowered to its first position. In this case, the secondstructure preferably includes a lid which acts to close the chamber inthe first position and the underside of which carries the holding means.Lid closure helps to prevent evaporation of volatile treatment liquids.

The second structure may be rotatably moveable around a central verticalaxis, enabling specimens to be loaded onto and unloaded from the secondstructure at a chosen position alongside the apparatus, either by arobotic arm or a human hand.

The apparatus may have the facility to change the liquid when in thefirst position, enabling the specimens to be treated by differentliquids in a succession of treatment stages, whilst retaining thechamber closed. For example, the apparatus may have a pump to fill andempty the chamber with a succession of chosen liquids which, in the caseof tissue specimens, may act to wash or otherwise treat the specimensprior to the specimens being imaged by means of optical projectiontomography.

According to another aspect of the invention there is provided a methodof treating tissue specimens by immersion in a liquid in a chamber, themethod comprising loading the specimens onto a holder so that thespecimens depend from the holder and are disposed above the liquid inthe chamber, effecting relative movement between the chamber and thespecimens in one direction to cause immersion of the specimens in theliquid whilst maintaining the chamber closed and protected from theenvironment during immersion, effecting relative movement between thechamber and the specimens in the opposite direction to bring thespecimens out of the liquid, and unloading the treated specimens fromthe holder.

The specimens may be treated by different liquids in a plurality oftreatment stages which are preferably carried out by successive emptyingand filling of the chamber with the different liquids, whilst thespecimens remain in the chamber and whilst the chamber remains closedand protected from the environment.

According to another aspect of the present invention, there is provideda method of performing any one or more of the analyses or procedureslisted hereunder comprising use of a method or apparatus of any of theaspects set out above.

There is also provided use of a method or apparatus as described in anyof the aspects as set out above in any one or more of the analyses orprocedures listed hereunder.

According to the present invention, the analyses and procedures of thepresent invention include:

Analysis of the structure of biological tissues.

Analysis of the function of biological tissues.

Analysis of the shapes of biological tissues.

Analysis of the distribution of cell types within biological tissues.

Analysis of the distribution of gene activity within biological tissues,including the distribution of:

-   -   RNA transcripts    -   proteins

Analysis of the distribution of transgenic gene activity withinbiological tissues,

Analysis of the distribution of cell activities within biologicaltissues,

including:

-   -   Cell cycle status including arrest    -   Cell death    -   Cell proliferation    -   Cell migration

Analysis of the distribution of physiological states within biologicaltissues.

Analysis of the results of immunohistochemistry staining techniques.

Analysis of the results of in-situ hybridisation staining techniques.

Analysis of the distribution of molecular markers within biologicaltissues, including any coloured or light-absorbing substances, such as:

-   -   5,5′-dibromo-4,4′-dichloro-indigo (or other halogenated indigo        compounds) formazan    -   or other coloured precipitates generated through the catalytic        activity of enzymes including: b-galactosidase, alkaline        phosphatase or other coloured precipitates formed upon catalytic        conversion of staining substrates,    -   including: Fast Red, Vector Red    -   And including any light-emitting substances,    -   Therefore including any fluorescent substances,    -   such as: Alexa dyes, FITC, rhodamine,    -   And including any luminescent substances,    -   such as green fluorescent protein (GFP) or similar proteins,    -   And including any phosphorescent substances.

Analysis of tissues from all plant species.

Analysis of any tissue for agricultural research,

including:

-   -   basic research into all aspects of plant biology (genetics,        development, physiology, pathology etc.)    -   analysis of tissues which have been genetically altered.

Analysis of tissues from all animal species.

including:

-   -   invertebrates    -   nematode worms    -   vertebrates    -   all types of fish (including teleosts, such as zebrafish, and        chondrycthes including sharks)    -   amphibians (including the genus Xenopus and axolotls)    -   reptiles    -   birds (including chickens and quails)    -   all mammals (including all rodents, dogs, cats and all primates,        including human)

Analysis of embryonic tissues for any purpose,

including:

-   -   research into any stem cell population    -   research into developmental biology    -   research into the causes of abnormal embryo development,        including human syndromes    -   autopsies of human terminated pregnancies (both spontaneous and        induced terminations)

Analysis of any tissues for the purpose of genomics research, including:

-   -   the analysis of any tissues for the purpose of genomics        research,    -   including:        -   the analysis of transgenic, knock-in, knock-down or            knock-out organisms the analysis or discovery of the            expression (or activity) of genes including their spatial            distribution, and their levels of expression        -   the analysis of discovery of abnormalities in the structure            or morphology of tissues, as a result of interference due to            wilful experimentation (such as genetic or physical            modifications including a chemical or biochemical genomics            approach), and/or spontaneous abnormalities (such as            naturally-occurring mutations)

Analysis of any tissue for the purpose of neurobiology research,

including:

-   -   the analysis of the morphology of nerves    -   the analysis of the pathways and connectivity of nerves    -   the analysis of parts of, or whole, animal brains

Analysis of any tissue for pharmaceutical research,

including:

-   -   the analysis of pharmaceutical substances (such as drugs,        molecules, proteins, antibodies),    -   including their spatial distribution within the tissue, and        their concentrations    -   the analysis or discovery of abnormalities in the structure or        morphology of tissues.

Analysis of tissues for medical research,

including:

-   -   research into the genetics, development, physiology, structure        and function of animal tissues    -   analysis of diseased tissue to further our understanding of all        types of diseases    -   including:    -   congenital diseases    -   acquired diseases        -   including:        -   infectious        -   neoplastic        -   vascular        -   inflammatory        -   traumatic        -   metabolic        -   endocrine        -   degenerative        -   drug-related        -   iatrogenic or        -   idiopathic diseases

Analysis of tissues for medical diagnosis, treatment or monitoring,including:

-   -   the diagnosis of cancer patients    -   including:        -   searching for cancerous cells and tissues within biopsies        -   searching for abnormal structure or morphology of tissues            within biopsies    -   the analysis of all biopsies    -   including the analysis of:        -   lymph nodes        -   polyps        -   liver biopsies        -   kidney biopsies        -   prostate biopsies        -   muscle biopsies        -   brain tissue    -   the analysis of tissue removed in the process of extracting a        tumour from a patient    -   including:        -   determining whether all the tumour has been removed        -   determining the type of tumour, and the type of cancer.

According to the present invention, samples for use in the presentinvention may be prepared as described in the earlier patentapplications and/or employing conventional pathological and histologicaltechniques and procedures well known to persons skilled in the art.

For example, in-situ hybridisation (particularly useful for detectingRNAs):Hammond K L, Hanson I M, Brown A G, Lettice L A, Hill R E“Mammalian and Drosophila dachsund genes are related to the Skiproto-oncongene and are expressed in eye and limb”. Mech Dev. 1998June;74(1-2):121-31.

Immunohistochemistry (particularly useful for detecting proteins andother molecules): Sharpe J, Ahlgren U, Perry P, Hill B, Ross A,Hecksher-Sorensen J, Baldock R, Davidson D. “Optical projectiontomography as a tool for 3D microscopy and gene expression studies”Science. 2002 Apr. 19;296(5567):541-5.

It will be appreciated that modification may be made to the inventionwithout departing from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described, by way of example, withreference to the accompanying drawings, in which:—

FIG. 1 is an isometric view of apparatus according to the invention andin an open condition,

FIG. 2 is an isometric view of the apparatus in a closed condition,

FIG. 3 is a fragmentary view, to an enlarged scale, of part of theapparatus in an open condition, showing specimens held by the apparatus,

FIG. 4 is a cross-sectional view of the apparatus,

FIG. 5 shows the apparatus in combination with a robotic arm,

FIG. 6 is an enlarged view of part of FIG. 5, and

FIG. 7 shows the robotic arm positioning a specimen onto the rotarystage of an OPT scanner.

DETAILED DESCRIPTION

The apparatus comprises a fixed structure 1 having a cylindrical outercasing 2 in the top of which is positioned an annular trough 3 which isopen at the top. Mounted in the fixed structure is a moveable structure4 having a central hub or spindle 5 on the top of which is mounted adisc-like lid 6 the underside of which carries a number of angularlyspaced and downwardly depending cylindrical magnets 7. The hub orspindle 5 is rotatably mounted in the fixed structure for rotation ofthe moveable structure about a central vertical axis indicated at 8 inFIG. 4. Also, the hub or spindle 5 is capable of vertical translationalmovement with respect to the fixed structure 1 along the vertical axis8.

Within the casing 2 are located motors and gearing for driving thestructure 4 both in rotation and translational movement, as indicated at9 in FIG. 4. The trough 3 is capable of being filled with liquids, andthe structure 1 includes containers for holding these liquids and pumpsfor filling and emptying the trough, as indicated at 10 in FIG. 4.

The magnets 7 are used to hold, in a detachable manner, tissue specimens12 each of which has been prepared with a metal mount 13 at one end ofthe specimen. This allows each metal 13 mount to depend from one of themagnets 7, with the specimen 12 depending downwardly from the mount 13.When the apparatus is in the open condition (FIG. 1), the magnets 7 areraised clear of the top of the trough 3 so that the specimens 12 can beattached to or removed from the magnets 7. When the apparatus is in theclosed position (FIG. 2), the lid 6 engages the top of the casing 2 andthe specimens 12 are immersed in a liquid 14 in the trough 3.

The robotic arm 15 shown in FIGS. 6 and 7 is used to load untreatedspecimens into the apparatus and also to transfer treated specimens fromthe apparatus to the rotary stage 16 of an OPT scanner where thespecimens are imaged.

In use, the robotic arm 15 is used to load specimens into the apparatus,each specimen being attached to the lower end of a corresponding magnet7 by virtue of the magnetic attraction between the magnet 7 and themetal mount 13 at one end of the specimen 12, the moveable structure 4being in the open position and being indexed in a rotational sense asthe specimens are loaded. When loaded with specimens, the moveablestructure is moved to its lowered or closed position, thereby immersingthe specimens 12 in the liquid 14 which has been pumped into the trough3. In this closed position, the lid 6 engages the upper rim of the outercasing 2 so that the liquid 14 is closed to the air, thereby allowingthe use of a volatile liquid, unlike known apparatus which uses conveyorbelts for transferring specimens through a liquid.

If it is required to treat the specimens by a succession of liquids, thefirst liquid is drained from the trough 3 and a second liquid pumpedthereinto, without the need for the trough 3 to be opened to the air.Moreover, the attachment of the metal mounts 13 to the magnets 7 retainsthe specimens 12 in their hanging positions so that the specimens do notengage the bottom of the trough 3, which could damage them.

Any number of treatment stages can be carried out in this manner, theliquid being changed without the need to move the specimens andemploying only a small volume of each treatment liquid.

After treatment of the specimens 12, the moveable structure 4 is raisedto its upper position and the treated specimens 12 are then transferredto the rotary stage 16 by means of the robotic arm 15, the structure 4being rotationally indexed to enable the robotic arm 15 to unload eachspecimen 12 in turn and to transfer the treated specimen 5 to the rotarystage 16.

Examples of liquids for treating the specimens are fixatives (such asparaformaldehyde or formalin), alcohols (in particular methanol andethanol) and organic solvents for clearing the specimens (in particularbenzyl alcohol and benzyl benzoate).

The apparatus and methods can be used in various analyses andprocedures, as set out below:

Analysis of the structure of biological tissues.

Analysis of the function of biological tissues.

Analysis of the shapes of biological tissues.

Analysis of the distribution of cell types within biological tissues.

Analysis of the distribution of gene activity within biological tissues,

including the distribution of:

-   -   RNA transcripts    -   proteins

Analysis of the distribution of transgenic gene activity withinbiological tissues,

Analysis of the distribution of cell activities within biologicaltissues,

including:

-   -   Cell cycle status including arrest    -   Cell death    -   Cell proliferation    -   Cell migration

Analysis of the distribution of physiological states within biologicaltissues.

Analysis of the results of immunohistochemistry staining techniques.

Analysis of the results of in-situ hybridisation staining techniques.

Analysis of the distribution of molecular markers within biologicaltissues,

including any coloured or light-absorbing substances,

such as:

-   -   5,5′-dibromo-4,4′-dichloro-indigo (or other halogenated indigo        compounds) formazan    -   or other coloured precipitates generated through the catalytic        activity of enzymes including: b-galactosidase, alkaline        phosphatase or other coloured precipitates formed upon catalytic        conversion of staining substrates, including: Fast Red, Vector        Red    -   And including any light-emitting substances,    -   Therefore including any fluorescent substances,    -   such as: Alexa dyes, FITC, rhodamine,    -   And including any luminescent substances,    -   such as green fluorescent protein (GFP) or similar proteins,    -   And including any phosphorescent substances.

Analysis of tissues from all plant species.

Analysis of any tissue for agricultural research,

including:

-   -   basic research into all aspects of plant biology (genetics,        development, physiology, pathology etc.)    -   analysis of tissues which have been genetically altered.

Analysis of tissues from all animal species,

-   -   including:    -   invertebrates    -   nematode worms    -   vertebrates    -   all types of fish    -   (including teleosts, such as zebrafish, and chondrycthes        including sharks)    -   amphibians (including the genus Xenopus and axolotls)    -   reptiles    -   birds (including chickens and quails)    -   all mammals (including all rodents, dogs, cats and all primates,        including human)

Analysis of embryonic tissues for any purpose,

including:

-   -   research into any stem cell population    -   research into developmental biology    -   research into the causes of abnormal embryo development,        including human syndromes    -   autopsies of human terminated pregnancies (both spontaneous and        induced terminations)

Analysis of any tissues for the purpose of genomics research,

including:

-   -   the analysis of transgenic, knock-in, knock-down or knock-out        organisms    -   the analysis or discovery of the expression (or activity) of        genes including their spatial distribution, and their levels of        expression    -   the analysis of discovery of abnormalities in the structure or        morphology of tissues,    -   as a result of interference due to wilful experimentation (such        as genetic or physical modifications including a chemical or        biochemical genomics approach),    -   and/or spontaneous abnormalities (such as naturally-occurring        mutations)

Analysis of any tissue for the purpose of neurobiology research,

including:

-   -   the analysis of the morphology of nerves    -   the analysis of the pathways and connectivity of nerves    -   the analysis of parts of, or whole, animal brains

Analysis of any tissue for pharmaceutical research,

including:

-   -   the analysis of pharmaceutical substances (such as drugs,        molecules, proteins, antibodies),    -   including their spatial distribution within the tissue, and        their concentrations    -   the analysis or discovery of abnormalities in the structure or        morphology of tissues.

Analysis of tissues for medical research,

including:

-   -   research into the genetics, development, physiology, structure        and function of animal tissues    -   analysis of diseased tissue to further our understanding of all        types of diseases    -   including:    -   congenital diseases    -   acquired diseases        -   including:        -   infectious        -   neoplastic        -   vascular        -   inflammatory        -   traumatic        -   metabolic        -   endocrine        -   degenerative        -   drug-related        -   iatrogenic or        -   idiopathic diseases

Analysis of tissues for medical diagnosis, treatment or monitoring,

including:

-   -   the diagnosis of cancer patients        -   including:        -   searching for cancerous cells and tissues within biopsies        -   searching for abnormal structure or morphology of tissues            within biopsies    -   the analysis of all biopsies    -   including the analysis of:        -   lymph nodes        -   polyps        -   liver biopsies        -   kidney biopsies        -   prostate biopsies        -   muscle biopsies        -   brain tissue    -   the analysis of tissue removed in the process of extracting a        tumour from a patient    -   including:        -   determining whether all the tumour has been removed        -   determining the type of tumour, and the type of cancer.

It will be appreciated that modification may be made to the inventionwithout departing from the scope of the invention.

1. Apparatus for treating tissue specimens by immersion in a liquid, theapparatus comprising a first structure providing a chamber for holdingthe liquid, and a second structure including holding means forreleasably holding the specimens, the first and second structures beingrelatively moveable in a direction having a vertical component between afirst position in which the holding means are relatively close to thechamber and in which the second structure closes the top of the chamberto enable the specimens to be immersed in the liquid whilst the latteris protected from the environment, and a second position in which theholding means are relatively distant from the chamber to enable thespecimens to be loaded onto or unloaded from the holding means. 2.Apparatus according to claim 1, wherein the first structure isstationary and the second structure is shiftable vertically with respectto the first structure.
 3. Apparatus according to claim 1 or 2, whereinthe holding means include magnets to enable specimens, each providedwith a metal mount, to be detachably retained on the second structure bymagnetic attraction.
 4. Apparatus according to any of the precedingclaims, wherein the chamber is in the form of an annular trough. 5.Apparatus according to claim 4, wherein the holding means hold thespecimens so that the latter depend from the holding means at angularlyspaced positions around a circle such that the specimens are loweredinto the trough as the second structure is lowered to its firstposition.
 6. Apparatus according to claim 4 or 5, wherein the secondstructure includes a lid which acts to close the chamber in the firstposition and the underside of which carries the holding means. 7.Apparatus according to any of the preceding claims, wherein the secondstructure is rotatably moveable around a central vertical axis, enablingspecimens to be loaded onto and unloaded from the second structure at achosen position alongside the apparatus, either by a robotic arm or ahuman hand.
 8. Apparatus according to any of the preceding claims,wherein the apparatus has the facility to change the liquid when in thefirst position, enabling the specimens to be treated by differentliquids in a succession of treatment stages, whilst retaining thechamber closed.
 9. A method of treating tissue specimens by immersion ina liquid in a chamber, the method comprising loading the specimens ontoa holder so that the specimens depend from the holder and are disposedabove the liquid in the chamber, effecting relative movement between thechamber and the specimens in one direction to cause immersion of thespecimens in the liquid whilst maintaining the chamber closed andprotected from the environment during immersion, effecting relativemovement between the chamber and the specimens in the opposite directionto bring the specimens out of the liquid, and unloading the treatedspecimens from the holder.
 10. A method according to claim 9, whereinthe specimens are treated by different liquids in a plurality oftreatment stages.
 11. A method according to claim 10, wherein thetreatment stages are carried out by successive emptying and filling ofthe chamber with the different liquids, whilst the specimens remain inthe chamber and whilst the chamber remains closed and protected from theenvironment.
 12. (canceled)
 13. (canceled)